czernobill

Hello,
thanks for your suggestions!

@Xabi
The addition of Tween is essential since there is no detectable growth without. The isolated bacteria are Corynebacteria. This genus includes several lipophilic species which can be cultured by the addition of Tween-80. For C. jeikeium it has been shown that it lacks  type I fatty acid synthase genes which is responsible for the synthesis of a large variety of fatty acids in  C.  ammoniagenes. Lipids in Corynebacteria are typically of straight chain saturated and monounsaturated and they are needed to form corynemycolic acids that are attached to the cell wall to establish a permeability barrier. Tween-80 represents a monounsaturated fatty acid and is widely used for the cultivation of Corynebacteria.

@Phil
It will be hard to find a bacteria free source but I could try including the lipid in the agar then autoclaving and test if my indicator strains will grow. If so, I could follow your recommendation.
One prolblem I will be likely to encounter is that my indicator strains will dominate and overgrow the isolated Corynebacteria. The latter are fastidious and slow growing and I normally incubate for 3 days for well isolated single colonies.
I have never done spot inoculation before, so it would be nice if you stated your opinion.

What maybe works is cutting and removing a small piece of the lipid mixture-containing agar and filling this hole with the same type of agar of ~45°C which has been inoculated with the Corynebacteria. Afterwards, I incubate for two days and the bacteria will hopefully grow inside the agar. Then, I inoculate the entire plate with the indicator strain and incubate for another one day checking for a zone of inhibition around the cut agar piece.

What do you think?

oh, I tested E. coli XL1-Blue MRF' (Stratagene) but I could not find any information on the strain's motility.
This week, I will spot a small volume of liquid inoculated broth on 0.1% agar containing plates, incubate and see what happens.

Hi,
yes, I did everything the same way.
16S was completely the same for all isolates. Then, I sequenced gyrA and rpoB but the sequences were again 100% identical. Okay, if your samples from Australia and China did not show any differences for ITS, this might also be the case for my isolates.

I will conduct ERIC fingerprinting, maybe yielding bands of different length. What do you think of this approach?


Another question concerning the identification of new species:
I read a lot of papers and the authors only sequenced the 16S rRNA gene. In addition, they conducted a lot of biochemical tests and also cell wall murein analysis and lipid analysis. Are the latter two essential when describing a new species from Corynebacterium genus?

The method papers mentioned in the publications are very old. For example, they use paper chromatography and to be honest, it will be very hard for me to reproduce the methods since I have never done this before.

Can I replace these analyses by sequence data of 4 genes instead of only one (16S rRNA)? I will add results from API Coryne but I have no idea about cell wall and lipid analysis.

Thank you!

Hi,
thanks for your suggestions!
Before storing the bacteria on plate I tested LB with vs without Tween. On plates lacking the substance there was no growth. There is is quite a large number of publications where they used Tween as additive for culturing Corynebacteria.

Quote

I am a little worried about taking the entire remnant from the plate. If this will not succeed, I would have nothing left. Until now, I have been trying to take about 10 colonies and inoculate a plate or liquid medium. This has not been working.

I followed Xabis advice and put the original plates to 37°C for three days and again inoculated a plate. Now, I am waiting for any colonies.
I emoved the original plates from the incubator and instead of putting it to 4°C, I put it into a jar inside my drawer.